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Abstract

Pigeon protozoal encephalitis (PPE) is an emerging central-nervous disease of domestic
pigeons (Columba livia f. domestica) reported in Germany and the United States. It is caused by the apicomplexan parasite
Sarcocystis calchasi which is transmitted by Accipter hawks. In contrast to other members of the Apicomplexa such as Toxoplasma and Plasmodium, the knowledge about the pathophysiology and host manipulation of Sarcocystis is scarce and almost nothing is known about PPE. Here we show by mRNA expression
profiling a significant down-modulation of the interleukin (IL)-12/IL-18/interferon
(IFN)-γ axis in the brains of experimentally infected pigeons during the schizogonic
phase of disease. Concomitantly, no cellular immune response was observed in histopathology
while immunohistochemistry and nested PCR detected S. calchasi. In contrast, in the late central-nervous phase, IFN-γ and tumor necrosis factor
(TNF) α-related cytokines were significantly up-modulated, which correlated with a
prominent MHC-II protein expression in areas of mononuclear cell infiltration and
necrosis. The mononuclear cell fraction was mainly composed of T-lymphocytes, fewer
macrophages and B-lymphocytes. Surprisingly, the severity and composition of the immune
cell response appears unrelated to the infectious dose, although the severity and
onset of the central nervous signs clearly was dose-dependent. We identified no or
only very few tissue cysts by immunohistochemistry in pigeons with severe encephalitis
of which one pigeon repeatedly remained negative by PCR despite severe lesions. Taken
together, these observations may suggest an immune evasion strategy of S. calchasi during the early phase and a delayed-type hypersensitivity reaction as cause of the
extensive cerebral lesions during the late neurological phase of disease.

Introduction

Sarcocystis calchasi is an apicomplexan parasite and the causative agent of pigeon protozoal encephalitis
(PPE), an emerging neurological disease of the domestic pigeon (Columba livia f. domestica) [1,2]. The definitive hosts of S. calchasi are Accipiter hawks of which the European subspecies of the Northern goshawk (Accipiter g. gentilis) has been experimentally identified to shed large quantities of infectious sporocysts
[3,4]. So far the domestic pigeon is the only identified intermediate host of the parasite.
Pigeons show a biphasic disease with polyuria, diarrhea and apathy during the schizogonic
first phase and severe central nervous signs such as torticollis and opisthotonus
associated with severe brain lesions about eight weeks post infection. At the same
time mature tissue cysts were present in skeletal muscles. The severity and onset
of central nervous signs clearly were dose dependent. In contrast, the intensities
of histopathologic lesions and immune cell infiltration in the brains appear to be
independent of the amount of administered sporocysts (102-105 per oral dose) [5]. Moreover, no intralesional parasitic stages were observed in H&E histopathology.
It therefore appears that the immune system of the pigeon is incapable of preventing
infection and an immunopathological basis of the central-nervous lesions has been
hypothesized [3]. In this context it can be speculated that S. calchasi may benefit from the induction of central-nervous malfunctioning and immobilization
as they may influence the rate of parasite’s transmission to its definitive host similar
as it is proposed for related apicomplexan parasites [6].

Several avian Sarcocystis spp. have been reported to induce central nervous signs (see [1] for overview). Encephalitis is most often reported to be associated with the schizont
stage of the parasite’s development. One notable example is Sarcocystis neurona. This parasite which is closely related to S. calchasi and most probably of avian origin is capable of inducing a central nervous disease
in a broad range of avian and mammalian species such as horses, cats, and dogs [7-10]. In many cases and even in extensive lesions the number of intralesional S. neurona merozoites and schizonts can be very low. It has been proposed that an immune response
triggered by cytokines and metabolites of the parasite may cause the extensive lesions
[11]. Recently the presence of S. neurona tissue cysts together with schizonts and merozoites has been confirmed for the first
time in southern sea otters (Enhydra lutris nereis) with encephalitis [12]. More significantly, ovine Sarcocystis spp. such as Sarcocystis tenella has been found capable of inducing a widespread encephalomyelitis associated with
degenerating tissue cysts and prominent central nervous signs [13,14].

Until now the biology of the hosts’ immune response against Sarcocystis spp. in general has only scarcely been addressed and whether this genus of parasites
may manipulate the immune response similarly to other Apicomplexa is unknown. However,
in vitro results suggest that S. neurona might be capable of down-modulating the IFN-γ signaling pathway [15,16]. It has therefore been proposed that Sarcocystis spp. may use similar evasion strategies than Toxoplasma gondii, a well-studied apicomplexan parasite that interferes with the IFN-γ signaling pathway
[17,18]. Here, we aimed at investigating the immune response and pathophysiology of PPE due
to S. calchasi during the schizogonic and late chronic phase of disease associated with central-nervous
signs. In most cases we confirmed the presence of parasitic stages in the brains of
the pigeons by immunohistochemistry and nested PCR. The cytokine expression profile
together with the morphological results of this study may suggest an immune evasion
strategy of the parasite that interferes with the Th1 response in the first phase
of the disease, while an overstimulated T-cell mediated immune response appears to
be characteristic for the second phase of the disease.

Material and methods

Samples of pigeons

The samples used for the present study originate from an experimental infection study
of S. calchasi in domestic pigeons [3]. All experiments were performed under governmental approval (No. Reg 0111/08). The
pigeons were orally inoculated with a range of 102 to 3 × 106 sporocysts shed by an experimentally infected Northern goshawk (A. g. gentilis) as described previously [5]. The pigeons depicted a biphasic disease. Two animal groups were established for
the purpose of this study. The eight pigeons of group A inoculated with 8 × 104 to 3 × 106 sporocysts deceased 7–12 dpi during the schizogonic early phase of disease. The five
pigeons of group B inoculated with 102 to 104 sporocysts were euthanized 51–65 dpi in the central nervous late phase of disease.
Two additional pigeons (L60, L74) were inoculated with 103 sporocysts, euthanized in the second phase at day 53 and 59 dpi and integrated into
group B (Table 1). Five uninfected pigeons were used as reference animals. The brains of all pigeons
were removed immediately after death [5]. One half was snap frozen and stored at −80°C until further use. The other half was
fixed in 4% neutral-buffered formaldehyde and embedded in paraffin 24 h later. In
addition, tissue samples from lung, heart, liver, spleen, kidneys, gizzard and skeletal
muscles were taken and processed equally.

Table 1.Numbers of immunohistochemically detected schizonts and tissue cysts in five consecutive
transversal section of brains of pigeons during first (group A) and second, central
nervous phase of disease (group B)

Antibodies against S. calchasi

S. calchasi sporocysts derived from a Northern goshawk euthanized 14 days after oral infection
were used for generation of S. calchasi-specific polyclonal antibodies [3]. Washed sporocysts were pretreated with 5% sodium hypochlorite for 30 min, washed
and resuspended in 15 mL Roswell Park Memorial Institute medium (RMPI) medium supplemented
with 10% fetal bovine serum (FBS) and 15% bovine bile and were incubated for 1 h at
37°C. Vero cells grown in RMPI medium were inoculated with 108 excysted sporozoites and supplemented with FBS, 10 000 IU/mL penicillin and 10 000
μg/mL dihydrostreptomycin. Merozoites were harvested after 12 days by rinsing the
monolayer with 10 mL 4°C Hank’s buffered salt solution, washed and resuspended in
2.5 mL PBS and passed through a PD-10 desalting column (GE Healthcare, Freiburg, Germany)
as previously described [19]. Purified merozoites were incubated in 4% formalin for 30 min and washed three times
in PBS. Standard immunization of two rabbits was conducted with 1 × 107 merozoites each. Histological sections of skeletal muscle infested with tissue cysts
and livers infested with merozoites and schizonts of S. calchasi from experimentally infected domestic pigeons were used to assess the specificity
of the serum.

Histopathology and immunohistochemistry

Formalin-fixed paraffin-embedded tissue was sectioned at 4 μm, mounted on glass slides
and stained with haematoxylin and eosin (H&E).

Immunohistochemistry was used to analyze the prevalence of parasitic stages of S. calchasi and expression of MHC-II, CD3 for T-cells and Pax-5 for B-cells in pigeon brains.
Serial sections of frozen brain samples were cut at 4 μm, mounted on adhesive glass
slides and were fixed in acetone for 10 min and dried for 20 min. Avidin-biotin blocking
of the cryostat sections was performed according to the manufacturer’s protocol (Dako
North America, Inc., Carpinteria, CA, USA). The slides were washed in PBS containing
0.05% Triton X-100 and blocked with PBS containing 2% BSA and 20% normal goat serum
for 30 min. Finally the sections were incubated with mouse-anti-chicken MHC-II specific
antibody 2G11 (1:50) for 1 h. The antibody 2G11 has been shown to cross-react with
MHC-II of multiple avian and non-avian species [20]. A goat anti-mouse IgG (1:200, Vector Laboratories, Burlingame, CA, USA) was used
as secondary antibody. MHC-II immunoreaction was visualized by incubating in ABC solution,
followed by HistoGreen-staining (Linaris, Wertheim-Bettingen, Germany) for 4 min at
room temperature.

Nested and quantitative real time (RT) PCR

S. calchasi DNA was detected by nested PCR targeting the ITS1 region as described previously
[4].

For candidate reference genes, three primer pairs for beta-actin, beta glucuronidase
(GUSB) and hydroxymethylbilane synthetase (HMBS) were used (A. Meyer, unpublished
observations). In addition, two new primer pairs for ribosomal protein L13 (RPL13)
and transferrin receptor protein (TFRC) were designed using Netprimer software (PREMIER
Biosoft, Palo Alto, CA, USA) based on a comparative sequence analysis of published
mRNA sequences of the domestic chicken (G. gallus f. dom.) and the zebra finch (Taeniopygia guttata) using MEGA4 (Table 2) [22]. In the same way a novel set of primers was designed for 10 cytokines of the domestic
pigeon including interleukin (IL) 1, IL-6, IL-7, IL-12, IL-15, IL-18, interferon gamma
(IFN-γ), transforming growth factor beta 2 (TGF-β2), LPS-induced TNF-α factor (LITAF),
TNF-like ligand 1A (TL1A) and the chemokine IL-8 (Tab. 2). For RT-PCR, the 15 μL reaction
mix included 10 μL Brilliant SYBR Green QPCR Master Mix (Applied Biosystems, Carlsbad,
CA) with 300 nM of each primer and 5 μL sample cDNA. Cycling conditions were 10 min
at 95°C followed by 40 cycles at 30 s at 95°C, 1 min at 58°C and 30 s at 72°C. Reference
genes were evaluated to quantify relative cytokine mRNA expression levels. RT-PCR
and data were analyzed using the MX 3000P Quantitative PCR System and MX Pro software
(Agilent). Each sample was analyzed in triplicate. Nuclease-free water was used as
negative controls in each run. Initially, primer efficiencies were determined and
primers with an efficiency below 90.0 excluded from further analysis (Table 1). Specificity of amplicons was evaluated against chicken sequences derived from GenBank
using MEGA5 (Table 1) and by melting curve analysis. Relative cytokine mRNA expression levels were normalized
by the efficiency-corrected ΔΔCt method against the expression of the three most stable
reference genes determined by the GeNorm algorithm [23-25]. Data are presented as fold change (FC) in cytokine expression levels in pigeons
of group A and B, respectively, normalized to the reference genes and relative to
the group of reference animals. Cut off values were set at > 2.0 for increased and
< 0.5 for reduced gene expression.

Table 2.Sequences of primers used for RT-qPCR and uncorrected (p) sequence distance of obtained pigeon amplicons to chicken mRNA

Statistical analysis

Gene expression data were analyzed with the Mann–Whitney-U test using SPSS software,
version 20.0 (SPSS, X). Results were considered statistically significant at p < 0.05 between two groups of animals.

Results

Histopathology and immunohistochemistry

Pigeons of group A that died during the schizogonic first phase of disease had no
histopathological lesions in the brain. No inflammatory cell response was discernible
in any area. Pigeons of group B were euthanized in the neurological second phase of
disease. All pigeons showing central nervous signs uniformly had a severe multifocal
lymphohistocytic and necrotizing encephalitis with prominent perivascular cuffing
(Figure 1) [5]. No pathological lesions were found in the reference animals.

Immunohistochemical testing revealed that the anti-merozoite antibody is capable of
labelling S. calchasi merozoites, schizonts and tissue cysts containing bradyzoites (data not shown). A
positive labelling for S. calchasi was observed in the cerebrum of pigeons of group A and B. Four of eight pigeons of
group A had rare schizonts in the neuropil (Figure 2 and Table 1). Mean schizont dimensions were 11.6 × 9.8 μm (n = 6, range = 7.3-13.4 × 6.5-13.2 μm). Three of seven pigeons of group B had few,
randomly distributed tissue cysts located in areas of the neuropil not associated
with pathological lesions or inflammatory cell reactions (Figure 3 and Table 1). The size of tissue cysts was in mean 19.0 × 15.1 μm (n = 14, range = 12.1-26.2 × 10.6-24.8 μm). All reference animals were negative for
S. calchasi by immunohistochemistry.

Figure 2.Immunohistochemical labelling of a schizont with polyclonal antiserum to S. calchasi in the brain of pigeon no. 7 during the first phase of disease. No associated cellular immune reaction or necrosis is discernible. In total, three
schizonts were identified in consecutive section of the brain of this pigeon. Bar,
30 μm.

Figure 3.Immunohistochemical labelling of a tissue cyst with polyclonal antiserum to S. calchasi in the brain of pigeon no. L60 during the second phase of disease. No associated pathological lesions can be seen. In total, four tissue cysts were identified
in the neuropil of this pigeon. Bar, 50 μm.

Areas with cerebral lesions including necrosis and gliosis as well as areas with mononuclear
cell infiltration had strong MHC-II labelling in all pigeons of group B (Figure 4). Most mononuclear cells were strongly CD3 positive (T-lymphocytes) while only few
cells were positive for Pax-5 (B-lymphocytes; Figure 5).

Figure 4.Immunohistochemical detection of MHC-II of a pigeon with cerebral lesions in the second
phase of disease. Most prominent signaling is discernable in areas of perivasular cuffing of mononuclear
cells (left) and areas of necrosis and gliosis (right). Bar, 300 μm.

Molecular detection of S. calchasi

The nested PCR amplified DNA specific for S. calchasi in the brains of all pigeons of group A and all but one (pigeon no. 14) of group
B (Figure 6). Reference animals were all negative. Processing controls and no template control
also gave negative results.

Figure 6.PCR detection of S. calchasi DNA in brains of pigeons with specific nested primer pairs targeting the ITS1 region.
All brains of pigeons of group A (no. 2–9) and pigeons of group B (no. 10–12, 15,
L60 and L74) except for no. 14 were positive. Five reference animals (r1-r5) used
for RT-qPCR as well as negative controls and two processing controls were negative.

Cytokine expression profile

Most stable reference genes for the cerebrum of pigeons of this study calculated by
the GeNorm algorithm were RPL13, HMBS and beta-actin. For group A, mRNA expression
levels of the Th-1 cytokines IL-12, IL-18 and IFN-γ were significantly down-modulated
while the proinflammatory cytokines IL-1, IL-6, IL-15 and the chemokine IL-8 were
significantly up-modulated when compared to the reference animals (Figure 7A). No regulation in mRNA expression levels was measured for TL1A, LITAF, IL-7 and
TGF-β2. In contrast, for group B IFN-γ expression was significantly up-modulated while
IL-18 was down-modulated and IL-12 was not regulated (Figure 7B). Furthermore, TL1A, LITAF and IL-7 were significantly up-modulated. IL-8 remained
up-modulated, while IL-1 and IL-15 were unregulated when compared with the reference
pigeons. IL-6 and TGF-β2 were also unregulated, but without statistical significance.

Discussion

As demonstrated previously, S. calchasi is capable of causing a severe biphasic central nervous disease in the domestic pigeon
[3]. Histologically, a severe lymphohistiocytic and necrotizing encephalitis was found
in the late neurological phase of disease. So far, intralesional stages of the parasite
had not been confirmed in the pigeon’s brains [5]. Because in tissue sections of cerebral sarcosporidiosis the parasitic load can be
low or difficult to detect despite extensive pathological lesions [3,11], we generated and established an anti-merozoite antiserum against S. calchasi. The antibody reliably detected merozoites, schizonts and tissue cysts including
bradyzoites by immunohistochemical analysis. Hereby we demonstrate that S. calchasi stages - although only very few - are present in about half of the brains in both
clinical phases of PPE. The presence of S. calchasi DNA could be confirmed by PCR results for the ITS1 region from the cerebrum in all
but one pigeon. Together the results indicate that S. calchasi is present in the brains of pigeons with PPE in both disease phases and may suggest
a direct involvement of the parasite in the development of the cerebral lesions. However,
since no direct association of parasitic stages with histopathological lesions was
detected, an unknown immunopathological mechanism may trigger the extensive inflammatory
lesions. This notion is underlined by one pigeon infected with 102 sporocysts with a strong cellular immune response but negative results for parasite
protein and DNA in the brain. To verify this, further experimental studies are needed
since it cannot be ruled out that at the onset of central nervous signs all parasites
are effectively eliminated by the immune system.

To further clarify the effect of S. calchasi on the cerebral immune response we established a novel panel of primers to measure
the expression level of 11 key immune effector genes and 5 reference genes by RT-qPCR.
We characterized an anti-parasite response profile of the host immune system. Most
notably, during the schizogonic, first phase of disease the important Th1 cytokines
IL-12, IL-18 and IFN-γ as well as IL-15 were significantly down-modulated. During
this phase, schizonts were present in various organs, most prominently in the liver,
spleen and endothelial cells [5]. In the brains, only very few schizonts were detected in the neuropil without discernible
lesions or immune cell infiltrations, although the expression of the major pro-inflammatory
cytokines IL-1 and IL-6 and the chemokine IL-8 were significantly up-modulated. This
may suggest that similar to other members of the Apicomplexa, S. calchasi is capable of manipulating the IL-12/IL-18/IFN-γ axis to evade the cellular immune
response [17].

Compared to closely related members of the Apicomplexa such as Toxoplasma gondii and Neospora caninum very little is known about the host immune response against Sarcocystis infection. IFN-γ has been shown to be essential for the protection against S. neurona neurological disease in mice [26]. IFN-γ is produced by T-cells, natural killer (NK) cells, monocytes and microglia
in the brain [27]. While IFN-γ KO mice show severe neurological disease after experimental infection
with S. neurona, SCID mice, which still have functional IFN-γ producing NK cells, only develop disease
after treatment with neutralizing anti-IFN-γ antibodies [26]. Furthermore, it has been shown that CD8+ T-cells are critical for the protection
from meningoencephalitis in C57BL/6 mice [28], while a humoral immune response seems to play no major role [29]. There is also first evidence that S. neurona may be capable of interfering with the cytokine signaling of the Th1 immune response.
IFN-γ production was reduced in lymphocytes extracted from Equine protozoal meningoencephalitis
(EPM) positive horses [16]. Isolated peripheral blood lymphocytes from horses with EPM that were co-cultured
with SnSAG1 produced significantly less IFN-γ after 48 h [15] and the cell-mediated immune responses to SnSAG1 were significantly reduced in horses
with EPM [16]. Together with the results of this study it is plausible to assume that Sarcocystis spp. in general may exhibit an immune evasion strategy that disrupts IFN-γ signaling.
Since the Th1-biased immune response is of major importance in clearing infection
with T. gondii and N. caninum [30,31], a balanced immune-modulation by the parasite is crucial for survival in its host.
Impairment of the IL-12 and IFN-γ expression is therefore one central immune evasion
strategy of T. gondii[17,18]. Furthermore, an impaired synthesis of IL-15 reduces the expression of IFN-γ which
enhances the survival of T. gondii[32]. IL-15 also activates CD4+ and CD8+ T-cells, of which CD8+ T cells are stimulated
to produce IFN-γ [33]. Interestingly, besides IL-12, IL-18 and IFN-γ, IL-15 was significantly down-modulated
in the first phase of S. calchasi infection. This temporary immune suppression may, similar to T. gondii[30], facilitate S. calchasi to infect host cells and to replicate in the absence of a protective immune response.

In contrast to the first phase, the second neurological phase of disease is associated
with a massive mononuclear cell infiltration and characterized by a markedly up-modulated
IFN-γ expression. Notably, IL-12 and IL-18 as important inducers of IFN-γ were not
up-modulated. IFN-γ is the key cytokine for the activation of mononuclear cells. This
correlates well with the prominence of T-cells, the granulomatous character of the
lesions and prominent MHC-II signaling in the pigeon brains. Chicken IFN-γ increases
the expression of class II MHC on antigen presenting cells and directly activates
macrophages and natural killer cells which can excrete further inflammatory effector
molecules such as TNF-α that may destroy tissue. [34-36]. Since TNF-α has not been identified in the avian genome, we tested TL1A and LITAF
which both were significantly up-modulated. Taken together this suggests an extensive
Th1-biased T-cell driven immune response, which appears inappropriate in the light
of a very low or absent parasite load.

Pigeons infected with S. calchasi depict neurological signs such as incoordination about eight weeks after infection
when tissue cysts mainly located in skeletal muscles contain infectious bradyzoites
[5]. This alteration in intermediate host behavior may lead to an increased predation
rate by the final host, the Northern goshawk. In contrast to pigeons, several natural
and aberrant hosts of avian Sarcocystis spp. (i.e. S. neurona) depict neurological signs associated with encephalitis only during the schizogonic
phase which does not allow transmission to a final host [7,9,37,38]. Changes in intermediate host behavior as reported for common voles (Microtus arvalis) and lemmings (Dicrostonyx richardsoni) infected by birds of prey-transmitted Sarcocystis cernae and Sarcocystis rauschorum, respectively, have been suggested to enhance parasite transmission due to increased
predation rates [39,40]. However, whether the change in behavior of pigeons induced by S. calchasi may be regarded as a parasite’s adaptation to enhance its fitness or simply as side
effect due to a delayed-type hypersensitivity reaction requires further investigation
to meet the manipulation hypothesis [41].

In conclusion, the observations of this study suggest that the Th-1 immune response
during the schizogonic phase of the S. calchasi development is down-modulated in the intermediate host. The absence of a strong host’s
cellular immune response in the pigeon may facilitate parasite evasion during acute
disease and subsequent formation of tissue cysts. The results of this study further
suggest that during the late central nervous phase of PPE a T-cell mediated delayed-type
hypersensitivity reaction may cause the cerebral lesions.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

Conceived and designed the experiments: PO, AM, ML, AG. Performed the experiments:
PO, AM. Analyzed the data: PO, AM, RK, BK. Contributed reagents/ materials/ analysis
tools: PO, ML, BK, AG. Wrote the paper: PO. All authors have read and approved the
final manuscript.